Capillary electrophoretic development of aptamers for a glycosylated VEGF peptide fragment.

The emergence of functional genomics and proteomics has added to the growing need for improved analysis methods that can detect and distinguish between protein variants resulting from allelic variation, mutation, or post-translational modification. Aptamers, single-stranded DNA or RNA molecules that fold into three-dimensional structures conducive to binding targets, have become an attractive alternative to antibodies for this type of analysis. Although aptamers have been developed for a wide range of target species, very few sequences have been identified that bind selectively to proteins with specific post-translational modifications. Using capillary electrophoresis-based selection, we have developed DNA aptamer sequences that selectively bind an N-glycosylated peptide fragment of vascular endothelial growth factor (VEGF). The selection method incorporates alternating positive- and counter-selection steps in free solution in order to obtain aptamers with both high affinity toward the glycosylated target and high selectivity versus a non-glycosylated variant. Affinity capillary electrophoresis and surface plasmon resonance binding assays indicate these sequences have low-µM dissociation constants and preferentially bind the glycosylated peptide with as much as 50-fold specificity. Such aptamers could serve as tools for rapid and simple monitoring of disease-linked functional changes in proteins, with potential applications in drug screening and disease diagnosis.